Engineered microorganisms &amp; methods for improved crotyl alcohol production

ABSTRACT

Disclosed are methods and engineered microorganisms that enhance or improve the production of crotyl alcohol. The engineered microorganisms include genetic modifications in alcohol dehydrogenase, alkene reductase or both enzymatic activities. By such genetic modifications, a crotyl alcohol production pathway is provided or improved.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/145,290 filed Apr. 9, 2015, the disclosure ofwhich is incorporated in its entirety herein by reference.

The entire contents of the ASCII text file entitled GN00025WO SequenceListing_ST25 created on Mar. 29, 2016 and having a size of 31 kilobytesis incorporated herein by reference.

BACKGROUND

Crotyl alcohol, also referred to as crotonyl alcohol, crotonol orbut-2-en-1-ol, is an unsaturated alcohol of the formula C₄H₈O and shownin FIG. 1. Crotyl alcohol is a valuable chemical intermediate serving asa precursor to alkenes including 1,3-butadiene, crotyl halides, esters,and ethers, which in turn are chemical intermediates in the productionof polymers, fine chemicals, agricultural chemicals, andpharmaceuticals.

Engineered microorganisms that produce increased crotyl alcohol areattractive alternatives to petroleum-based productions. But when suchalcohols are produced by the cells and released they can accumulate intothe media during culturing. Changes in the culture media conditions canaffect a microorganism's growth and viability because microbes, such asbacteria, generally have low alcohol tolerance.

Alcohol removal from the media during culturing is one way to addressthis problem. But such removal processes can be expensive andinefficient. Another approach is to use natural bacterial isolates thatdemonstrate tolerance to higher alcohol levels. Such strains, however,are generally not suitable for industrial production or engineering.

SUMMARY OF THE INVENTION

Disclosed are methods and engineered microorganisms that produce crotylalcohol as a bioproduct or as an intermediate for the production ofother bioproducts.

In one embodiment, a method is provided for producing a bioproduct usingan engineered microorganism, where crotyl alcohol is the bioproduct, orcrotyl alcohol is an intermediate in a bioproduct pathway (e.g.1,3-butadiene, 1,3-butanediol, methyl vinyl carbinol), the methodcomprising:

-   -   culturing the engineered microorganism under conditions        resulting in crotyl alcohol production or use of crotyl alcohol        as the intermediate in a bioproduct pathway resulting in        bioproduct production, wherein the engineered microorganism        comprises at least one genetic modification causing partial or        complete loss of activity in (a) an alcohol dehydrogenase (ADH)        that converts crotyl alcohol to crotonaldehyde, (b) an ADH that        converts butyraldehyde to butanol, or (c) an alkene reductase        that converts crotonaldehyde to butyraldehyde or any combination        of (a), (b) and (c).

In some embodiments, the microorganism further comprises at least oneexogenous nucleic acid encoding an enzyme of a bioproduct pathway, wherethe bioproduct pathway is a crotyl alcohol pathway or uses a crotylalcohol as an intermediate in the bioproduct pathway.

In another embodiment, an engineered microorganism is disclosedcomprising at least one genetic modification causing partial or completeloss of activity in (a) an alcohol dehydrogenase (ADH) that convertscrotyl alcohol to crotonaldehyde, (b) an ADH that converts butyraldehydeto butanol, or (c) an alkene reductase that converts crotonaldehyde tobutyraldehyde or any combination of (a), (b) and (c) and at least oneexogenous nucleic acid encoding an enzyme of a bioproduct pathway, wherethe bioproduct pathway is a crotyl alcohol pathway or uses crotylalcohol as an intermediate in the bioproduct pathway.

In still other embodiments, a method is provided for producing abioproduct using an engineered microorganism, where crotyl alcohol isthe bioproduct, or crotyl alcohol is an intermediate in a bioproductpathway (e.g. 1,3-butadiene, 1,3-butanediol, methyl vinyl carbinol), themethod comprising:

-   -   providing an engineered microorganism having at least one        genetic modification causing partial or complete loss of        activity in in (a) an alcohol dehydrogenase (ADH) that converts        crotyl alcohol to crotonaldehyde, (b) an ADH that converts        butyraldehyde to butanol, or (c) an alkene reductase that        converts crotonaldehyde to butyraldehyde or any combination of        (a), (b) and (c); and    -   culturing the engineered microorganism under conditions        resulting in crotyl alcohol production or use of crotyl alcohol        as the intermediate in a bioproduct pathway resulting in        bioproduct production.

In some embodiments, the alcohol dehydrogenases and alkene reductasessubject to the partial or complete loss of activity are native to themicroorganism and the alcohol dehydrogenase is capable of convertingcrotyl alcohol to crotonaldehyde and/or converting butyraldehyde tobutanol, and the alkene reductase is capable of convertingcrotonaldehyde to butyraldehyde. These native activities have beenidentified as undesirable activities when they compete with desirableenzyme activities that comprise a pathway to crotyl alcohol or a crotylalcohol-derived bioproduct.

The microorganism can be further genetically modified to increaseactivity of an enzyme converting crotonaldehyde to crotyl alcohol, e.g.,a crotonaldehyde reductase (alcohol forming), particularly wherecrotonaldehyde is a by-product and not a bioproduct pathwayintermediate.

Such disclosed methods and engineered microorganisms allow for growth oncrotyl alcohol levels higher than microorganisms without the geneticmodifications. Such microorganisms can also produce significantly morecrotyl alcohol. In addition, the disclosed methods and microorganismscan increase the efficiency of crotyl alcohol yields and/or decreaseprocess costs by reducing or eliminating crotyl alcohol into undesirableor wasteful pathways.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. shows exemplary pathways for production of crotyl alcohol andbutadiene from acetyl-CoA, glutaconyl-CoA, glutaryl-CoA,3-aminobutyryl-CoA or 4-hydroxybutyryl-CoA via crotyl alcohol. Enzymesfor transformation of the identified substrates to products include: A.acetyl-CoA:acetyl-CoA acyltransferase, B. acetoacetyl-CoA reductase, C.3-hydroxybutyryl-CoA dehydratase, D. crotonyl-CoA reductase (aldehydeforming), E. crotonaldehyde reductase (alcohol forming), F. crotylalcohol kinase, G. 2-butenyl-4-phosphate kinase, H. butadiene synthase,I. crotonyl-CoA hydrolase, synthetase, transferase, J. crotonatereductase, K. crotonyl-CoA reductase (alcohol forming), L.glutaconyl-CoA decarboxylase, M., glutaryl-CoA dehydrogenase, N.3-aminobutyryl-CoA deaminase, O. 4-hydroxybutyryl-CoA dehydratase, P.crotyl alcohol diphospholdnase.

FIG. 2 shows exemplary pathways for converting crotyl alcohol(2-buten-1-ol) to 3-buten-2-ol and/or butadiene. Enzymes are A. crotylalcohol kinase, B. 2-butenyl-4-phosphate kinase, C. butadiene synthase,D. 3-buten-2-ol synthase, E. 3-buten-2-ol synthase, F. crotyl alcoholisomerase, a vinylisomerase, G. 3-buten-2-ol dehydratase, H. crotylalcohol dehydratase, a dehydratase/vinylisomerase, I. crotyl alcoholdiphosphokinase, J. butadiene synthase (from 2-butenyl-4-phosphate).Crotyl alcohol can be chemically dehydrated to butadiene.

FIG. 3 shows reactions for conversion of crotyl alcohol to by-products(Step C, G and/or H) and for conversion of a by-product crotonaldehydeto crotyl alcohol (Step B). The enzymes for transforming the identifiedsubstrates include, B. crotonaldehyde reductase (alcohol forming), alsoreferred to as an aldehyde reductase (alcohol forming), C. crotylalcohol dehydrogenase, G. crotonaldehyde reductase (also referred to asan alkene reductase), H. butyraldehyde reductase (alcohol forming).

FIGS. 4A-B show growth curves of E. coli strain K-12 substr. M01655 andvarious modifications grown on glucose (FIG. 4A) and on glucose+crotylalcohol (FIG. 4B) in terms of culture turbidity at OD420-580 relative toculture time.

FIGS. 5A-B show growth curves of E. coli strains ATCC 8739 and variousmodifications grown on glucose (FIG. 5A) and grown on glucose 4-crotylalcohol (FIG. 5B) in terms of culture turbidity at OD420-580 relative toculture time.

FIGS. 6A-D show crotyl alcohol and butanol measurements in various nemAand yghD deleted strains (3-GGGF, 3-GHDO, 3-HAEF, 3-HAEG).

DETAILED DESCRIPTION

The embodiments of the invention described here are not intended to beexhaustive or to limit the description to the precise forms disclosed inthe following detailed description. Rather, the embodiments are chosenand described so that others skilled in the art can appreciate andunderstand the principles and practices of the description.

All publications and patents mentioned here are hereby incorporated byreference in their entirety. The publications and patents disclosed hereare provided solely for their disclosure.

The term “alcohol dehydrogenase” (also referred to as ADH) as used hererefers to enzymes that can convert crotyl alcohol to crotonaldehydeand/or butyraldehyde to butanol, and includes those classified as EC1.1.1. An enzyme converting crotyl alcohol to crotonaldehyde is alsoreferred to here as a crotyl alcohol dehydrogenase (crotyl ADH). Analcohol dehydrogenase converting butyraldehyde to butanol is alsoreferred to here as a butyraldehyde reductase (alcohol forming).

The term “alkene reductase” refers to an enzyme that can convertcrotonaldehyde to butyraldehyde.

The term “bioproduction”, as used here refers to microbial synthesis.The terms “production” and “biosynthesis” are used interchangeably whenreferring to microbial synthesis of a desired or referenced substance orproduct.

The term “engineered” refers to a microbial organism or microorganismhas at least one genetic modification not normally found in a naturallyoccurring strain, wild-type strain or the parental host strain of thereferenced species.

The term “exogenous” as it is used here refers to the referencedmolecule or the referenced activity that is introduced into a hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity or bioproduct, the term refers to an activity that isintroduced into the parental host organism. The source can be, forexample, a homologous or heterologous encoding nucleic acid thatexpresses the referenced activity following introduction into theparental host microbial organism. Accordingly, exogenous includesgenetic modification to a regulatory region of a native gene or to aregulatory protein of a native gene that results in expression of thatnative gene or its encoded enzyme that is different than in the absenceof that modification. Therefore, the term “endogenous” refers to areferenced molecule or activity that is present in the host. Similarly,the term when used in reference to expression of an encoding nucleicacid refers to expression of an encoding nucleic acid contained withinthe microbial organism. The term “heterologous” refers to a molecule oractivity derived from a source other than the referenced species whereas“homologous” refers to a molecule or activity derived from the hostmicrobial organism. Accordingly, exogenous expression of a disclosedencoding nucleic acid can utilize either or both a heterologous orhomologous encoding nucleic acid.

The term “genetic modifications” as used here refers to an alteration inwhich expression of nucleic acids, polypeptides that are deleted orintroduced to encode or express metabolic polypeptides, enzymes, othernucleic acid additions, nucleic acid deletions and/or other functionaldisruption of the microbial organism's genetic material. Suchmodifications for example may be made in, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. The term“genetic alteration” is used interchangeably with genetic modification.

The term “metabolic modification” refers to a biochemical reaction thatis altered from its naturally occurring state. Therefore, engineeredmicroorganisms can have genetic modifications to nucleic acids encodingmetabolic polypeptides, or functional fragments thereof.

The terms “microbial,” “microbial organism” or “microorganism” refers toorganisms that exist as microscopic cells and encompass prokaryotic oreukaryotic cells or organisms having a microscopic size and includesbacteria, archaea and eubacteria of all species as well as eukaryoticmicroorganisms such as yeast and fungi. The term also includes cells ofany species that can be cultured for the production of a bioproduct.

The term “parental microbial organism,” or grammatical equivalentsthereof (and including parent host microorganism or host microorganism)when used in the context of a genetic modification, is understood tomean a parent microorganism or strain in which the particular geneticmodification has not been made but otherwise has the same geneticmakeup. For example, if a strain of microbial organism is used to make agenetic modification that increases expression of a gene product, theparent strain would be the starting strain into which the heterologousgene is introduced. Similarly, a parent microbial organism in which agenetic modification has been made to decrease expression, such as agene disruption or gene deletion, the parent microbial organism wouldhave the same genetic background except for the gene disruption ordeletion. However, it is understood that a parent microbial organism candiffer by more than one genetic modification, either gene additionand/or disruption, depending on whether a single or multiple geneticmodifications are being considered. One skilled in the art will readilyunderstand the meaning of such a parent microbial organism in that themicrobial organism is considered to be an appropriate control, asunderstood in the art, for observing the effect of one or more geneticmodifications.

The term, “partial loss,” “reduced activity,” and the like refers to amicroorganism's enzyme or an isolated enzyme that exhibits a loweractivity level than that measured in a parent microbial organism or itsnative enzyme. This term also can include elimination of that enzymaticactivity.

Disclosed are engineered microorganisms with genetic modifications toADH enzymatic activity, alkene reductase enzymatic activity or both.Such engineered microorganisms are capable of accumulating greatercrotyl alcohol amounts (e.g. titers), having greater availability ofcrotyl alcohol, as well as capable of growing on higher crotyl alcohollevels compared to a parental host strain or microorganisms without thedisclosed genetic modifications. Such engineered microorganisms may beused for the fermentation production of crotyl alcohol. The engineeredmicroorganisms may also be useful in providing improved crotyl alcoholpathways and other bioproduct pathways via crotyl alcohol as anintermediate. In addition, the disclosed methods and microorganisms canincrease the efficiency of crotyl alcohol yields and/or decrease processcosts by reducing or eliminating undesirable or wasteful pathways thatdivert crotyl alcohol into unwanted metabolic products or intermediates,some of which may adversely impact the microorganism's health. Forexample, by having greater crotyl alcohol availability compared to theparental strain, more crotyl alcohol can be enzymatically converted to abioproduct of interest, such as butadiene, 1,3-butanediol or methylvinyl carbinol or to an intermediate to a bioproduct of interest. Thecells are cultured in suitable medium using methods and media known inthe art.

Exemplary microorganisms that may serve as the parental host microbialorganism to be engineered include, for example, bacteria, yeast, fungusor any of a variety of other microorganisms applicable to fermentationprocesses, including methylotophs or microbes engineered to bemethylotrophs. Exemplary bacteria include the genus selected fromAcinetobacter, Escherichia, Klebsiella, Anaerobiospirillum,Actinobacillus, Castellaniella, Mannheimia, Bacillus, Zymomonas,Lactococcus, Lactobacillus, Streptomyces, Clostridium, Pseudomonas,Thauera and Zymomonas.

Other exemplary bacteria include species selected from Escherichia coli,Klebsiella oxytoca, Anaerobiospirillum succiniciproducens,Actinobacillus succinogenes, Castellaniella caeni, Castellanielladaejeonensis, Castellaniella defragrans, Castellaniella denitrificans,Castellaniella ginsengisoli, Castellaniella hirudinis, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Bacillusmethanolicus, Corynebacterium glutamicum, Gluconobacter oxydans,Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum,Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonasfluorescens, Thauera aminoaromatica, Thauera aromatics, Thauerabutanivorans, Thauera chlorobenzoica, Thauera humireducens, Thaueralinaloolentis, Thauera mechernichensis, Thauera phenylacetica, Thaueraselenatis, Thauera terpenica and Pseudomonas putida.

Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowia lipolytica, andthe like.

E. coli is a particularly useful parental host organism since it is awell characterized microbial organism suitable for genetic engineering.Other particularly useful host microorganisms include yeast such asSaccharomyces cerevisiae. It is understood that any suitablemicroorganism can be used to introduce metabolic and/or geneticmodifications to produce an engineered microorganism, which engineeredmicroorganism is capable of producing a desired bioproduct. In someembodiments, E. coli include strains E. coli Str K-12 substr. MG1655 andE. coli ATCC 8739 variant. In one embodiment the the host organism isnot Bacillus subtilis.

Alcohol Dehydrogenases

In some embodiments, the genes encoding ADH activity are deleted ordisrupted. The disclosed ADH enzymes targeted for gene deletion ordisruption can also include those ADH enzymes that use reducednicotinamide adenine dinucleotide (NADH) and/or NADPH as an electrondonor.

In some embodiments, increase in the amount of crotyl alcohol and/ortolerance to crotyl alcohol can be caused by the disruption of thedisclosed ADH genes that encode for enzymes which otherwise catalyzesthe conversion of crotyl alcohol to crotonaldehyde and/or butyraldehydeto butanol. Deletion in the ADH activity results in reduction or partialor full loss of ADH activity and as shown in FIG. 3 as enzyme C.Deletion of ADH (e.g. as shown as C and/or H) drives the process towardscrotyl alcohol. In some embodiments, the ADH enzymes to be deleted arenative or endogenous to the engineered microorganism. The nativeactivities may be identified as undesirable activities when they competewith desirable enzyme activities that include crotyl alcohol or crotylalcohol-derived bioproducts.

In some embodiments, the enzymes for converting crotyl alcohol tocrotonaldehyde and/or butyraldehyde to butanol are the same. In otherembodiments, the enzymes for converting crotyl alcohol to crotonaldehydeare different from the enzymes for converting butyraldehyde to butanol.

Table 1 lists exemplary ADH genes that can be disrupted or deleted.

TABLE 1 Gene GenBank ID GI Number Organism alrA BAB12273.1 9967138cinetobacter sp. Strain M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli bdh I NP_349892.115896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542Clostridium acetobutylicum adhA YP_162971.1 56552132 Zymomonas mobilisCbei_2181 YP_001309304.1 150017050 Clostridium beijerinckii NCIMB 8052

Other ADH genes that can be disrupted or deleted include ADH enzymesactive on allyl alcohols can also be suitable for oxidizing crotylalcohol to crotonaldehyde (see FIG. 3, enzyme C). An exemplary allylalcohol dehydrogenase is the NtRed-1 enzyme from Nicotiana tabacum(Matsushima et al, Bioorg. Chem. 36: 23-8 (2008)). A similar enzyme hasbeen characterized in Pseudomonas putida MB1 but the enzyme has not beenassociated with a gene to date (Malone et al, Appl. Environ. Microbiol65: 2622-30 (1999)). Yet another allyl alcohol dehydrogenase is thegeraniol dehydrogenase enzymes of Castellaniella defragrans,Carpoglyphus lactis and Ocimum basilicum (Lueddeke et al, AEM 78:2128-36(2012)). Alcohol dehydrogenase enzymes with broad substrate specificityare also applicable here, such as include alrA encoding a medium-chainalcohol dehydrogenase for C2-C14 (Tani et al., Appl. Environ. Microbiol.66:5231-5235 (2000)), yghD, yahK, adhE and fucO from E. coli(Sulzenbacher et al., J Mol Biol 342:489-502 (2004)), and bdh I and bdhII from C. acetobutylicum which converts butyryaldehyde into butanol(Walter et al, J. Bacteriol. 174:7149-7158 (1992)). YqhD of E. colicatalyzes the reduction of a wide range of aldehydes using NADPH as thecofactor, with a preference for chain lengths longer than C(3)(Sulzenbacher et al, J Mol Biol 342:489-502 (2004); Perez et al., JBiol. Chem. 283:7346-7353 (2008)). The adhA gene product from Zymomonasmobilis has been demonstrated to have activity on a number of aldehydesincluding formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde,and acrolein (Kinoshita et al., App. Microbiol. Biotechnol. 22:249-254(1985)).

Other ADH genes that can be disrupted or deleted include those listed inTable 2.

TABLE 2 GenBank Gene Accession No. GI No. Organism MDR BAM52497.1407959257 Synechocystis sp. PCC 6803 NT-RED1 BAA89423 6692816 Nicotianatabacum geoA CCF55024.1 372099287 Castellaniella defragrans GEDH1Q2KNL6.1 122200955 Ocimum basilicum GEDH BAG32342.1 188219500Carpoglyphus lactis alrA BAB12273.1 9967138 Acinetobacter sp. strain M-1ADH2 NP_014032.1 6323961 Saccharomyces cerevisiae fucO NP_417279.116130706 Escherichia coli yqhD NP_417484.1 16130909 Escherichia coliyahK P75691.1 2492774 Escherichia coli adhE NP_415757.1 16129202Escherichia coli bdh I NP_349892.1 15896543 Clostridium acetobutylicumbdh II NP_349891.1 15896542 Clostridium acetobutylicum adhA YP_162971.156552132 Zymomonas mobilis Bdh BAF45463.1 124221917 Clostridiumsaccharoperbutylacetonicum

Additional ADH enzymes that can be targeted for gene disruption ordeletion can be identified based on sequence homologies to the disclosedADH sequences. In some embodiments, the ADH enzymes include ADH enzymeshaving at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity tothe polypeptide or polynucleotide sequence of full length proteinsequences in SEQ ID NOs: 11-14, shown in Table 5. Exemplary deletionsare shown in SEQ ID NOs: 1-4.

The disclosed ADH enzymes can convert crotyl alcohol to crotonaldehydeand/or butyraldehyde to butanol. In some embodiments, geneticmodifications (e.g. deletions or disruptions) to ADHs cause partial orcomplete loss of ADH activity. In some embodiments, deletion of the ADHgenes or activity is deletions of the native genes or enzymes. In stillother embodiments, deletion of the native ADH genes encoding ADHactivity that can convert crotyl alcohol to crotonaldehyde and/orbutyraldehyde to butanol. In some embodiments, the deletions in adhgenes include deletions in at least one, two, three, four or five adhgenes. In some embodiments, the deletions are deletions in the Eco/l-encoded adh genes. In other embodiments, the deletions are to oneor more sequences as disclosed here as SEQ ID NOs: 1-4 and shown inTable 5.

ADH enzymes that may be modified include activity encoded by, forexample, the genes adhE, yqhD, yahK. In some embodiments, themicroorganisms may include deletions or disruptions in the adhE, adhP,yqhD, yahK, genes or combinations thereof. For example, the geneticmodifications may include genetic deletions to single genes and denotedas ΔadhE, ΔadhP, ΔyqhD and ΔyahK The delta symbol denotes a genedeletion. The deletions can include multiple deletions such as to morethan one deletion, two, three or four deletions. For example, twodeletions in the desired adh gene can be denoted as (ΔadhEΔadhP), (ΔadhEΔadhP), (ΔadhEΔyqhD), and (ΔadhEΔyahK); three deletions in the desiredadh genes are denoted as (ΔadhEΔadhPΔyqhD), (ΔadhEΔadhPΔyahK), and fourdeletions in the desired adh genes are denoted as(ΔadhEΔadhPΔyqhDΔyahK). Such genetic deletions to the disclosed adhgenes, polynucleotides and/or polypeptides result in a partial orcomplete loss of ADH activity.

In some embodiments, the partial or complete loss of activity is in ADHactivity capable of converting crotyl alcohol to crotonaldehyde and/orbutyraldehyde to butanol. The partial or complete loss of ADH activityallows the microorganism to grow on higher crotyl alcohol levels as wellas in some embodiments grow at a faster rate on crotyl alcohol comparedto the parental host microorganism or a microorganism without thegenetic modification. In other embodiments, the genetic modifications(e.g. deletions) to the adh genes allow the microorganism to accumulatehigher crotyl alcohol levels.

Methods for introducing the exogenous gene are described more fullybelow in reference to crotyl alcohol pathways and other pathways.

Alkene Reductase Activity

In some embodiments, genetic modifications (e.g. deletions) to alkenereductase can cause partial or complete loss of alkene reductaseactivity. The disclosed alkene reductase enzymes can also use reducednicotinamide adenine dinucleotide (NADH) and/or NADPH as an electrondonor.

In some embodiments, increase in the amount of crotyl alcohol ortolerance to crotyl alcohol can be caused by the disruption of thedisclosed alkene reductase which otherwise catalyzes the conversion ofcrotonaldehyde to butyraldehyde. Deletion in the alkene reductase generesults in reduction or partial or full loss of alkene reductaseactivity and as shown in FIG. 3 as enzyme G, driving the process towardscrotyl alcohol. This in turn eliminates crotyl alcohol from enteringother pathways such as into butanol via crotonaldehyde and as shown forexample in FIG. 3. In some embodiments, the alkene reductase enzymesdeleted are native or endogenous to the engineered microorganism. Thenative activities may be identified as undesirable activities when theycompete with desirable enzyme activities that include crotyl alcohol orcrotyl alcohol-derived bioproducts.

Also contemplated here are deletion/disruption of alkene reductaseenzyme-encoding genes that include 2-ene-al reductase or α, β-ene-alreductase activity, or enzymes that accept a wide range ofα,β-unsaturated alkenals. In other embodiments, the alkene reductaseactivity may also be reduced/eliminated by deletion of genes that encodethose enzymes that have N-ethylmaleimide reductase activity. It wassurprisingly found that the nemA gene product was active on 2-ene-alssuch as crotonaldehyde.

In some embodiments, the alkene reductase activity can also bereduced/eliminated by deletion of genes that encode enzymes from the E.coli K12 subgroup. In still other embodiments, the alkene reductase isfrom E coil str. K12 substr. MG1655 or E. coli ATCC 8739 variant. Instill other embodiments, the alkene reductase activity is enzyme ofsequence of GI: 732684441, SEQ ID NO: 15 of Table 5. In still otherembodiments, alkene reductase activity targeted for gene deletion ordisruption is encoded by SEQ ID. NO. 5 as shown in Table 5. In someembodiments, the disclosed alkene reductase activity targeted forreduced/elimination of activity may be encoded by the nemA gene from Ecoll. In some embodiments, the native or endogenous gene that canconvert crotonaldehyde to butyraldehyde is deleted in a microorganism.

Other embodiments include alkene reductase activity encoded by the ygjMgene from B. subtillis, NADH-dependent flavin oxidoreductase, or ithomologs or paralogs in other species, that may be targeted for genedisruption or deletion. Other bacterial homologs include PETN(pentaerythritol tetranitrate) reductase (French C. E., Nicklin S.,Bruce N. C. Sequence and properties of pentaerythritol tetranitratereductase from Enterobacter cloacae PB2, J. Bacteriol. 178:6623-6627,1996), GTN (glycerol trinitrate) reductase (Snape et al., Purification,properties, and sequence of glycerol trinitrate reductase fromAgrobacterium radiobacter. J. Bacteriol. 179:7796-7802, 1997), MR(morphinone reductase) (French et al., Bacterial morphinone reductase isrelated to Old Yellow Enzyme. Biochem. J. 312:671-678, 1995),2-cyclohexenone reductase (Rohde et al., Thermoregulated expression andcharacterization of an NAD(P)H-dependent 2-cyclohexen-1-one reductase inthe plant pathogenic bacterium Pseudomonas syringae pv. glycinea. J.Bacteriol. 181:814-822, 1999), and the xenobiotic reductases A and Bfrom Pseudomonas sp. (Blehert et al. Cloning and sequence analysis oftwo Pseudomonas flavoprotein xenobiotic reductases. J. Bacteriol.181:6254-6263, 1999).

Additional alkene reductase enzymes that can be targeted for genedisruption or deletion can be identified based on sequence homologies tothe disclosed nemA, ygjM and other sequences described here. In someembodiments, the alkene reductase having at least about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to the polypeptide or polynucleotidesequence of full length protein sequences of nemA, ygjM or the otheralkene reductase enzymes described here.

Accordingly, in some embodiments, the microorganisms may includedeletions or disruptions in the ΔnemA gene or ΔygjM gene or homologsthereof and combinations thereof (e.g. ΔnemAΔygjM). In otherembodiments, the ygjM gene may be from Bacillus subtilis. In otherembodiments, the nemA gene from E coil may be deleted. In still otherembodiments, the homologs or paralogs of the yjgM gene in E. coli may bedeleted; the homologs or paralogs of the nemA gene may be deleted fromBacillus subtilis or other species.

Such genetic deletions to the disclosed nemA and/or ygjM genes,polynucleotides and/or polypeptides result in a partial or complete lossof alkene reductase activity. The partial or complete loss of alkenereductase activity that results in crotonaldehyde to butyraldehydeallows the microorganism to grow on higher crotyl alcohol levels as wellas in some embodiments grow at a faster rate on crotyl alcohol comparedto the parental host microorganism or a microorganism without thegenetic modification. In other embodiments, the genetic modifications(e.g. deletions) to the alkene reductase genes or activity allow themicroorganism to accumulate higher crotyl alcohol levels.

Alkene reductase may also be reduced/eliminated in microorganism of thegenus Acetobacterium, Achromobacter, Acremonium, Agrobacterium,Alcaligenes, Bacillus, Bradyrhizobium, Burkholderia, Caloramator,Castellaniella, Cephalosporium, Clostridium, Escherichia, Eubacterium,Filifactor, Fusobacterium, Kluyveromyces, Mesorhizobium, Moorella,Ochrobactrum, Oxalophagus, Oxobacter, Paenibacillus, Pseudomonas,Ralstonia, Rhizobium, Rhodotorula, Salmonella, Shigella, Sinorhizobium,Sporohalobacter, Syntrophospora, Thauera, Thermoanaerobacter,Thermoanaerobacterium, Tilachlidium, Vibrio, Xanthobacter, or Yersinia.

Other, enzymes having α,β-enoate reductase activity include an enzymefrom Acremonium strictum CBS114157 (deposit date Dec. 19, 2003;deposited under the terms of the Budapest treaty), Clostridiumtyrobutyricum DSM1460 (available from the Deutsche SammlungMikroorganismen und Zellkulturen), Moorella thermoacetica DSM1974(available from the Deutsche Sammlung Mikroorganismen und Zellkulturen)(an enzyme which also under DSMI974 until Jan. 1, 1980, was namedClostridium thermoaceticum), Ochrobactrum anthropi NCIMB41200 (depositdate Dec. 16, 2003; deposited under the terms of the Budapest treaty),or Clostridium kluyveri DSM555 (available from the Deutsche SammlungMikroorganismen und Zellkulturen).

In some embodiments, the alkene reductase and the ADH genes are deleted.In some embodiments, the ADH genes adhE, adhP, yqhD, yahK, or variouscombinations thereof along with nemA are deleted. For example, theengineered microorganism may have genetic deletions in nemA and in asingle gene selected from adhE adhP, yqhD and yahK or multiple genesselected from (adhE, adhP), (adhE, adhP), or (adhE, yqhD), (adhE, yahK),(adhE, adhP, yqhD), (adhE, adhP, yahK), and (adhE, adhP, yghD, yahK).

In still other embodiments, the ADH genes adhE, adhP, yqhD, yahK, orvarious combinations thereof along with ygjM are deleted. For example,the engineered microorganism may have genetic deletions in ygjM and in asingle gene selected from adhE adhP, yqhD and yahK or multiple genesselected from (adhE, adhP), (adhE, adhP), or (adhEyqhD), (adhE, yahK),(adhE, adhP, yqhD), (adhE, adhP, yahK), and (adhE, adhP, yqhD, yahK).

in still other embodiments, the ADH genes adhE, adhP, yqhD, yahK, orvarious combinations thereof along with nemA are deleted in E. coli. Instill other embodiments, the ADH genes and homologs or paralogs of adhE,adhP, yqhD, yahK, or various combinations thereof along with ygjM aredeleted in Bacillus subtilis.

The disclosed genetic modifications that include gene disruptions orgene deletions that encode for ADH and/or alkene reductase enzymesinclude a deletion of the entire gene, deletion of portions of the gene,deletion or other modification of a regulatory sequence required fortranscription or translation, deletion of a portion of the gene whichresults in a truncated gene product (e.g., enzyme) or by any of variousmutation strategies that reduces activity (including to no detectableactivity level) of the encoded gene product. A disruption may broadlyinclude a deletion of all or part of the nucleic acid sequence encodingthe enzyme, and also includes, but is not limited to other types ofgenetic modifications, e.g., introduction of stop codons, frame shiftmutations, introduction or removal of portions of the gene, introductionof conditional sensitive mutations (e.g. temperature sensitive), andintroduction of a degradation signal, those genetic modificationsaffecting mRNA transcription levels and/or stability, altering thepromoter or repressor upstream of the gene encoding the enzyme andaltering mRNA translation, e.g. insertion of stem loop or removing orinactivating an RBS.

In various embodiments, genetic modifications can be to the DNA, mRNAencoded from the DNA, and the corresponding amino acid sequence thatresults in partial or complete loss of the polypeptide activity. Manydifferent methods can be used to make a cell having partial, reduced orcomplete loss of the polypeptide activity. For example, a cell can beengineered to have a disrupted regulatory sequence orpolypeptide-encoding sequence using common mutagenesis or knock-outtechnology. See, e.g., Datsenko and Wanner, Proc. Natl. Acad. Sci USA(2000), 97(12): 6640-6645, and Methods in Yeast Genetics (1997 edition),Adams et al., Cold Spring Harbor Press (1998), and Manual of IndustrialMicrobiology and Biotechnology (3^(rd) edition), edited by Richard H.Boltz, Arnold L Demain, Julian E. Davies.

One particularly useful method of gene disruption is complete genedeletion because it reduces or eliminates the occurrence of geneticreversions in the genetically modified microorganisms. Accordingly, adisruption of a gene whose product is an enzyme thereby disruptsenzymatic function. Alternatively, antisense technology can be used toreduce the activity of a particular polypeptide. For example, a cell canbe engineered to contain a cDNA that encodes an antisense molecule thatprevents a polypeptide from being translated. Gene silencing can also beused to reduce the activity of a particular polypeptide (see forexample, Metabolic Engineering of Escherichia coli Using Synthetic SmallRegulatory RNAs. Na D, Yoo S M, Chung H, Park H, Park J R, Lee S Y;Nature Biotechnology. 2013 Feb. 31(2):170-4).

Thus, in some embodiments, the partial reduction of activity isdecreased functional activity. The functional activity can be decreasedduring either or both the growth or stationary phases. As discussedreduced activity can be achieved for example by deleting a gene,decreasing gene expression, or decreasing the activity or availabilityof the polypeptide encoded by the gene. The gene can be modifieddirectly, e.g. use of a weaker promoter, one or more amino acidsubstitutions or deletions or insertions, or can be modulated bymodifying its regulatory gene for example.

The resultant genetic disruption or deletion can result in the partial,reduced or complete loss of enzymatic activity. In some embodiments, theenzymatic conversion of the indicated substrate(s) to indicatedproduct(s) under known standard conditions for that enzyme is at least10, at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, or at least 90 percent less than the enzymaticactivity for the same biochemical conversion by a native (non-modified)or parental host enzyme under a standard specified conditions.

A cell having reduced enzymatic activity can be identified using anymethod known in the art. For example, enzyme activity assays can be usedto identify cells having reduced enzyme activity, see, for example,Enzyme Nomenclature, Academic Press, Inc., New York 2007. Other assaysthat may be used to determine the reduction in ADH and alkene reductaseinclude GC/MS analysis. In other examples, levels of NADP/NADPH may bespectroscopically monitored. For example the NADH/NADPH may be monitoredcolorimetrically using NADP/NADPH assay kits (ab65349) available fromABCAM™.

Gene deletions may be accomplished by mutational gene deletionapproaches, and/or starting with a strain having reduced or noexpression of one or more of these enzymes, and/or other methods knownto those skilled in the art. Gene deletions may be effectuated by any ofa number of known specific methodologies, including but not limited tothe Red/ET methods.

The Red/ET recombination is known to those of ordinary skill in the artand described in Datsenko and Wanner, Proc. Natl. Acad. Sci USA (2000),97(12): 6640-6645. Other deletion methods include those described inU.S. Pat. Nos. 6,355,412 and 6,509,156, issued to Stewart et al. Each ofthese references is incorporated by reference here for its teachings ofthis method. Material and kits for such method are available from GeneBridges™ and the method may proceed by following the manufacturer'sinstructions. The method involves replacement of a target gene by aselectable marker via homologous recombination performed by therecombinase from X-phage. The host organism expressing k-red recombinaseis transformed with a linear DNA product coding for a selectable markerflanked by the terminal regions (generally ^(˜)50 bp, and alternativelyup to about ^(˜)300 bp) homologous with the target gene. The markercould then be removed by another recombination step performed by aplasmid vector carrying the FLP-recombinase, or another recombinase,such as Cre.

Targeted deletion of parts of microbial chromosomal DNA or the additionof foreign genetic material to microbial chromosomes may be practiced toalter a host cell's metabolism so as to reduce or eliminate productionof undesired metabolic products. This may be used in combination withother genetic modifications.

The microorganism can be further genetically modified to increaseactivity of an enzyme converting crotonaldehyde to crotyl alcohol (seefor example, E in FIG. 1 and B in FIG. 3). In some embodiments, theenzyme is a crotonaldehyde reductase (alcohol forming). Increase of acrotonaldehyde reductase (alcohol forming) is used particularly wherecrotonaldehyde is a by-product and not a bioproduct pathwayintermediate.

In some embodiments, some microorganisms include at least one exogenousnucleic acid encoding an enzyme that converts crotonaldehyde to crotylalcohol. In some embodiments, some microorganisms include at least oneexogenous nucleic acid encoding crotonaldehyde reductase (alcoholforming) introduced into the microorganism. In other embodiments, theexogenous nucleic acids that may be increased include those sequencesdisclosed as SEQ ED NOs: 1-4 and 11-14. In other embodiments, anengineered microorganism with a deletion in the disclosed adh and/oralkene reductase genes may also include at least one exogenous nucleicacid with ADH activity that can convert an aldehyde to an alcohol. Insome embodiments, the microorganism includes at least one exogenousnucleic acid of SEQ ID NO: 12 and 14. In other embodiments, themicroorganism expresses the gene yqhD and/or yahK.

Enzymes exhibiting crotonaldehyde reductase (alcohol forming) activityare capable of forming crotyl alcohol from crotonaldehyde. The followingenzymes can naturally possess this activity or can be engineered toexhibit this activity. Exemplary genes encoding enzymes that catalyzethe conversion of an aldehyde to alcohol (e.g., alcohol dehydrogenase orequivalently aldehyde reductase) include genes encoding a medium-chainalcohol dehydrogenase for C2-C14 (Tani et at, Appl. Environ. Microbiol.66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et al,Nature 451:86-89 (2008)), yqhD from E. coli which has preference formolecules longer than C(3) (Sulzenbacher et al., J. Mol. Biol.342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum whichconverts butyraldehyde into butanol (Walter et al, J. Bacteriol.174:7149-7158 (1992)). ADH1 from Zymomonas mobilis has been demonstratedto have activity on a number of aldehydes including formaldehyde,acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita,Appl. Microbiol. Biotechnol. 22:249-254 (1985)). Cbei_2181 fromClostridium beijerinckil NCIMB 8052 encodes yet another useful alcoholdehydrogenase capable of converting crotonaldehyde to crotyl alcohol.

TABLE 3 Protein GenBank ID GI Number Organism alrA BAB12273.1 9967138Acinetobacter sp. Strain M-1 ADH2 NP_014032.1 6323961 Saccharomycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli bdh I NP_349892.115896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542Clostridium acetobutylicum adhA YP_162971.1 56552132 Zymomonas mobilisCbei_2181 YP_001309304.1 150017050 Clostridium beijerinckii NCIMB 8052

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC1.1.1.61) also fall into this category. Such enzymes have beencharacterized in Ralstonia eutropha (Bravo et al., J. Forensic Sci.49:379-387 (2004)), Clostridium kluyveri (Wolff et al., Protein Expr.Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al, J.Biol. Chem. 278:41552-41556 (2003)).

TABLE 4 Protein GenBank ID GI Number Organism 4hbd YP_726053.1 113867564Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM555 4hbd Q94B07 75249805 Arabidopsis thaliana

Crotyl Alcohol Pathways

The disclosed engineered microorganisms with the gene deletions ordisruptions that cause the partial or full loss of ADH and/or alkenereductase activity may be used in the production of crotyl alcohol orother bioproducts that are produced using crotyl alcohol as anintermediate. Such disclosed engineered microorganisms may also includecrotyl alcohol pathways. Numerous microorganisms and enzymatic pathwaysto produce crotyl alcohol or use crotyl alcohol as an intermediate to abioproduct are known. For example, with reference to FIG. 1, theengineered microorganisms can have one of the following pathways: EF;ABCDE; ABCK; ABCIJE; LK, MK, NK, OK; LDE; MDE; NDE, ODE; WE, MIJE; NIJE;OIJE and each combination of any one or more of A, B, C, D, E, F, I, J,K, L, M, N and O. Details for the pathways to crotyl alcohol or forconversion of crotyl alcohol to butadiene described in FIG. 1 can befound, for example, in PCT International Application Publication No. WO2011/140171 titled Microorganisms and Methods for the Biosynthesis ofButadiene. In some embodiments, the engineered microorganism with adeletion in ADH, alkene reductase or both may include any one of more ofthe steps and enzymes shown in FIG. 1. Other exemplary pathways forcrotyl production include those described in PCT InternationalApplication Publication No. WO 2011/140171 titled Microorganisms andMethods for the Biosynthesis of Butadiene and in PCT InternationalApplication Publication No WO 2014/152434 titled Microorganisms andMethods for Producing Butadiene and Related Compounds by FormateAssimilation. Other exemplary pathways for crotyl alcohol productioninclude U.S. Application No. 62/020,901 filed Jul. 3, 2014 and U.S.Application No. 62/082,747 filed Nov. 21, 2014, both titledMicroorganisms for Producing 4C-5C Compounds with Unsaturation andMethods Related Thereto. Still other exemplary pathways to crotylalcohol or for conversion of crotyl alcohol to butadiene include PCTInternational Application Publication No. WO 2013/057194 titled Processfor the Enzymatic Production of Butadiene from Crotyl Alcohol and PCTInternational Application Publication No. WO 2013/188546 titled Methodsfor biosynthesizing 1,3 butadiene.

Each of these cited patents or patent applications are incorporatedherein by reference in their entirety.

Depending on the crotyl alcohol biosynthetic pathway constituents of aselected host microbial organism, the engineered microorganism caninclude at least one exogenously expressed crotyl alcoholpathway-encoding nucleic acid and up to all encoding nucleic acids forone or more crotyl alcohol biosynthetic pathways. For example, crotylalcohol biosynthesis can be established in a host deficient in a pathwayenzyme or protein through exogenous expression of the correspondingencoding nucleic acid. In a host deficient in all enzymes or proteins ofa crotyl alcohol pathway, exogenous expression of all enzyme or proteinsin the pathway can be included, although it is understood that allenzymes or proteins of a pathway can be expressed even if the hostcontains at least one of the pathway enzymes or proteins. For example,exogenous expression of all nucleic acids and/or polypeptides encodingenzymes or proteins in a pathway for production crotyl alcohol can beincluded, such as an acetyl-CoA:acetyl-CoA acyltransferase, anacetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, acrotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase(alcohol forming), a crotyl alcohol kinase, (FIG. 1, steps A-E).

Bioproducts Via Crotyl Alcohol

Once the crotyl alcohol is obtained or produced by the disclosedengineered microorganism and methods, the crotyl alcohol can beconverted to a desired bioproduct (e.g. butadiene, 1,3-butanediol,methyl vinyl carbinol). For example, the crotyl alcohol can be furtherused as an intermediate in the bioproduction of 3-Buten-2-ol (alsoreferred to as methyl vinyl carbinol (MVC)) and/or butadiene bybiosynthetic processes, chemical or enzymatic processes.

FIG. 1 and FIG. 2 provide exemplary pathways to butadiene from crotylalcohol. FIG. 2 provides an exemplary pathway to butadiene via one ormore of the following intermediates: 2-butenyl-4-phosphate,2-butenyl-4-diphosphate, and/or 3-buten-2-ol. Optionally, crotyl alcoholcan be introduced into this pathway via any crotyl alcohol-producingpathway known in the art, and including conversion from 3-buten-1-ol or3-buten-2-ol. In some embodiments, the crotyl alcohol is produced by apathway of FIG. 1 and then introduced into a pathway of FIG. 2.

With reference to FIG. 2, the engineered microbial organism can have anyone of the following pathways: D; DG; AD; ADG; E; EG; BE; BEG; ABE;ABEG; lE; IEG; F; FG; G; H; J; AJ; A; AB; ABC and each combination ofany one or more of steps D, E, F with any one or more of steps of FIG.2, where when at least one of A, B, C, G or H is present then (i) atleast one other novel step or pathway is present. Further details forthe pathways described in FIG. 2 are found, for example, in PCTInternational Application Publication No. WO 2011/140171, in PCTInternational Application Publication No WO 2014/152434 titledMicroorganisms and Methods for Producing Butadiene and Related Compoundsby Formate Assimilation, and in PCT International ApplicationPublication No. WO 2014/033129 titled Production of volatile dienes byenzymatic dehydration of light alkenols, U.S. Application No. 62/020,901filed Jul. 3, 2014 and U.S. Application No. 62/082,747 filed Nov. 21,2014, both titled Microorganisms for Producing 4C-5C Compounds withUnsaturation and Methods Related Thereto and in other referencesdescribed herein. For example, enzymes suitable for FIG. 2 Steps F, G orH can be in the EC 4.2.1 class, of which linalool dehydratase assignedto EC 4.2.1.127 or a variant thereof is of particular interest, Variantsof linalool dehydratase have been reported in PCT InternationalApplication Publication No. WO 2014/184345. Still other microorganismsand pathways can be found in PCT International Application PublicationNo. WO 2013/188546 titled Methods for biosynthesizing 1,3 butadiene.Thus a parental cell provided herein includes a microorganism comprisinga pathway to crotyl alcohol as shown in FIG. 1 and a dehydratase such asa linalool dehydratase.

Other exemplary pathways for the production of butadiene include thosedescribed in PCT International Application Publication No. WO2011/140171 titled Microorganisms and Methods for the Biosynthesis ofButadiene; and U.S. Application No. 62/020,901 filed Jul. 3, 2014 andU.S. Application No. 62/082,747 filed Nov. 21, 2014, both titledMicroorganisms for Producing 4C-5C Compounds with Unsaturation andMethods Related Thereto.

Sources of encoding nucleic acids for a butadiene, crotyl alcohol,3-buten-2-ol, pathway enzyme or protein can include, for example, anyspecies where the encoded gene product is capable of catalyzing thereferenced reaction. Such species include both prokaryotic andeukaryotic organisms including, but not limited to, bacteria, includingarchaea and eubacteria, and eukaryotes, including yeast, plant, insect,animal, and mammal, including human.

Exemplary species for such sources include, for example, Escherichiaspecies, including Escherichia coli, Escherichia fergusonii,Castellaniella defragrans, Thauera linaloolentis, Thauera terpenica,Methanocaldococcus jannaschii, Leptospira interrrogans, Geobactersulfiereducens, Chloroflexus aurantiacus, Roseijtexus sp. RS-1,Chloroflexus aggregans, Achromobacter xylosoxydans, Clostrdia species,including Clostridium kluyveri, Clostridium symbiosum, Clostridiumacetobutylicum, Clostridium saccharoperbutylacetonicum, Clostridiumljungdahlii, Trichomonas vaginalis G3, Trypanosoma brucei,Acidaminococcus fennentans, Fusobacterium species, includingFusobacterium nucleatum, Fusobacterium mortiferum, Corynebacteriumglutamicum, Rattus norvegicus, Homo sapiens, Saccharomyces species,including Saccharomyces cerevisiae, Apsergillus species, includingAspergillus terreus, Aspergillus oryzae, Aspergillus niger, Gibberellazeae, Pichia stipitis, Mycobacterium species, including Mycobacteriumsmegmatis, Mycobacterium avium, including subsp. pratuberculosis,Salinispora arenicola Pseudomonas species, including Pseudomonas sp.CF600, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonasaeruginosa, Ralstonia species, including Ralstonia eutropha, Ralstoniaeutropha iMP134, Ralstonia eutropha HI6, Ralstonia pickettii,Lactobacillus plantarum, Klebsiella oxytoca, Bacillus species, includingBacillus subtilis, Bacillus pumilus, Bacillus megaterium, Pedicoccuspentosaceus, Chlorofexus species, including Chloroflexus aurantiacus,Chloroflexus aggregans, Rhodobacter sphaeroides, Methanocaldococcusjannaschii, Leptospira interrrogans, Candida maltose, Salmonellaspecies, including Salmonella enterica serovar Typhimurium, Shewanellaspecies, including Shewanella oneidensis, Shewanella sp. MR-4,Alcaligenes faecalis, Geobacillus stearothermophilus, Serratiamarcescens, Vibrio cholerae, Eubacterium barkeri, Bacteroidescapillosus, Archaeoglobus fulgidus, Archaeoglobus fulgidus, Haloarculamarismortui, Pyrobaculum aerophilum str. IM2, Rhizobium species,including Rhizobium leguminosarum, as well as other exemplary speciesdisclosed here or available as source organisms for corresponding genes.

However, with the complete genome sequence available for now more than550 species (with more than half of these available on public databasessuch as the NCBI), including 395 microorganism genomes and a variety ofyeast, fungi, plant, and mammalian genomes, the identification of genesencoding the requisite butadiene, crotyl alcohol, 3-buten-2-ol,biosynthetic activity for one or more genes in related or distantspecies, including for example, homologues, orthologs, paralogs andnonorthologous gene displacements of known genes, and the interchange ofgenetic alterations between organisms is routine and well known in theart. Accordingly, the metabolic alterations allowing biosynthesis ofbutadiene, crotyl alcohol, 3-buten-2-ol described here with reference toa particular organism such as E. coli can be readily applied to othermicroorganisms, including prokaryotic and eukaryotic organisms alike.Given the teachings and guidance provided here, those skilled in the artwill know that a metabolic alteration exemplified in one organism can beapplied equally to other organisms.

In addition to direct fermentation to produce butadiene, crotyl alcoholor 3-buten-2-ol (methyl vinyl carbinol) can be separated, purified (forany use), and then dehydrated to butadiene in a second step involvingmetal-based catalysis, see for example FIG. 2. Suitable product pathwaysand enzymes, methods for screening and methods for isolating are foundin: WO2011140171A2 published 10 Nov. 2011 entitled Microorganisms andMethods for the Biosynthesis of Butadiene; WO2012018624A2 published 9Feb. 2012 entitled Microorganisms and Methods for the Biosynthesis ofAromatics, 2, 4-Pentadienoate and 1, 3-Butadiene; O2011140171A2published 10 Nov. 2011 entitled Microorganisms and Methods for theBiosynthesis of Butadiene; WO2013040383A1 published 21 Mar. 2013entitled Microorganisms and Methods for Producing Alkenes;WO2012177710A1 published 27 Dec. 2012 entitled Microorganisms forProducing Butadiene and Methods Related thereto; WO2012106516A1published 9 Aug. 2012 entitled Microorganisms and Methods for theBiosynthesis of Butadiene; WO2013028519A1 published 28 Feb. 2013entitled Microorganisms and Methods for Producing 2,4-Pentadienoate,Butadiene, Propylene, 1,3-Butanediol and Related Alcohols; and U.S. Ser.No. 61/799,255 filed 15 Mar. 2013.

The chemical process includes dehydration to butadiene. Alcoholdehydration is known in the art and can include various thermalprocesses, both catalyzed and non-catalyzed. In some embodiments, acatalyzed thermal dehydration employs a metal oxide catalyst or silica.In some embodiments, the process is performed by chemical dehydration inthe presence of a catalyst. For example, it has been indicated thatcrotyl alcohol can be dehydrated over bismuth molybdate (Adams, C. R. J.Catal. 10:355-361, 1968) to afford 1,3-butadiene (e.g. see FIG. 2).

Dehydration can be achieved via activation of the alcohol group andsubsequent elimination by standard elimination mechanisms such as E1 orE2 elimination. Activation can be achieved by way of conversion of thealcohol group to a halogen such as iodide, chloride, or bromide.

Activation can also be accomplished by way of a sulfonyl, phosphate orother activating functionality that convert the alcohol into a goodleaving group. In some embodiments, the activating group is a sulfate orsulfate ester selected from a tosylate, a mesylate, a nosylate, abrosylate, and a triflate. In some embodiments, the leaving group is aphosphate or phosphate ester. In some such embodiments, the dehydratingagent is phosphorus pentoxide.

In a typical process for converting crotyl alcohol into butadiene,crotyl alcohol is passed, either neat or in a solvent, and either inpresence or absence of steam, over a solid inorganic, organic ormetal-containing dehydration catalyst heated to temperatures in therange of 40-400° C. inside of the reaction vessel or tube, leading toelimination of water and release of butadiene as a gas, which iscondensed (butadiene bp=−4.4° C.) and collected in a reservoir forfurther processing, storage, or use. Typical catalysts can includebismuth molybdate, phosphate-phosphoric acid, cerium oxide, kaolin-ironoxide, kaolin-phosphoric acid, silica-alumina, and alumina. Typicalprocess throughputs are in the range of 0.1-20,000 kg/h. Typicalsolvents are toluene, heptane, octane, ethylbenzene, and xylene.

In other embodiments, butadiene can be produced by the enzymaticconversion of crotyl alcohol via crotyl phosphate or crotyl diphosphateand as disclosed in WO 2013/057194 or via enzymatic dehydration(isomerization/dehydration) by an enzyme of the EC 4.2.1 class,preferably a linalool dehydratase, for example as reported in PCTInternational Application Publication Nos. WO 2014/033129 or WO2014/184345.

Embodiments are described here with general reference to the metabolicreaction, reactant or product thereof, or with specific reference to oneor more nucleic acids or genes encoding an enzyme associated with orcatalyzing, or a protein associated with, the referenced metabolicreaction, reactant or product. Unless otherwise expressly stated here,those skilled in the art will understand that reference to a reactionalso constitutes reference to the reactants and products of thereaction. Similarly, unless otherwise expressly stated here, referenceto a reactant or product also references the reaction, and reference toany of these metabolic constituents also references the gene or genesencoding the enzymes that catalyze or proteins involved in thereferenced reaction, reactant or product. Likewise, given the well-knownfields of metabolic biochemistry, enzymology and genomics, referencehere to a gene or encoding nucleic acid also constitutes a reference tothe corresponding encoded enzyme and the reaction it catalyzes or aprotein associated with the reaction as well as the reactants andproducts of the reaction.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refer to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed here, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed here a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

The engineered microbial organisms can contain stable geneticalterations, which refers to microorganisms that can be cultured forgreater than five generations without loss of the genetic alteration.Generally, stable genetic alterations include modifications that persistgreater than 10 generations, persist more than about 25 generations, andgreater than 50 generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified here, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics andmolecular biology, those skilled in the art will readily be able toapply the teachings and guidance provided here to essentially all otherorganisms. For example, the E. coli metabolic alterations exemplifiedhere can readily be applied to other species by incorporating the sameor analogous encoding nucleic acid from species other than thereferenced species. Such genetic alterations include, for example,genetic alterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less than 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the engineeredmicroorganism. An example of orthologs exhibiting separable activitiesis where distinct activities have been separated into distinct geneproducts between two or more species or within a single species.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used here. Functional similarityrequires, for example, at least some structural similarity in the activesite or binding region of a nonorthologous gene product compared to agene encoding the function sought to be substituted. Therefore, anonorthologous gene includes, for example, a paralog or an unrelatedgene.

Therefore, in identifying and constructing the disclosed engineeredmicrobial organisms having biosynthetic capability, those skilled in theart will understand with applying the teaching and guidance providedhere to a particular species that the identification of metabolicmodifications can include identification and inclusion or inactivationof orthologs. To the extent that paralogs and/or nonorthologous genedisplacements are present in the referenced microorganism that encode anenzyme catalyzing a similar or substantially similar metabolic reaction,those skilled in the art also can utilize these evolutionally relatedgenes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well-knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.2.29+(Jan. 6 2014) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.2.29+(Jan. 6 2014) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

For various embodiments, the genetic modifications include variousgenetic manipulations, including those directed to change regulation of,and therefore ultimate activity of, an enzyme or enzymatic activity ofan enzyme identified in any of the disclosed pathways. Such geneticmodifications may be directed to transcriptional, translational, andpost-translational modifications that result in a change of enzymeactivity and/or selectivity under selected and/or identified cultureconditions and/or to provision of additional nucleic acid sequences suchas to increase copy number and/or mutants of an enzyme related to crotylalcohol production. Specific methodologies and approaches to achievesuch genetic modification are well-known to one skilled in the art, andinclude, but are not limited to increasing expression of an endogenousgenetic element; decreasing functionality of a repressor gene;introducing a heterologous genetic element; increasing copy number of anucleic acid sequence encoding a polypeptide catalyzing an enzymaticconversion step to produce crotyl alcohol; mutating a genetic element toprovide a mutated protein to increase specific enzymatic activity;over-expressing; under-expressing; over-expressing a chaperone; knockingout a protease; altering or modifying feedback inhibition; providing anenzyme variant having one or more of an impaired binding site for arepressor and/or competitive inhibitor; knocking out a repressor gene;evolution, selection and/or other approaches to improve mRNA stabilityas well as use of plasmids having an effective copy number and promotersto achieve an effective level of improvement. Random mutagenesis may bepracticed to provide genetic modifications that may fall into any ofthese or other stated approaches. The genetic modifications furtherbroadly fall into additions (including insertions), deletions (such asby a mutation) and substitutions of one or more nucleic acids in anucleic acid of interest. In various embodiments a genetic modificationresults in improved enzymatic specific activity and/or turnover numberof an enzyme. Without being limited, changes may be measured by one ormore of the following: K_(rn); K_(cat); and K_(cat)/K_(rn)=catalyticefficiency.

In one embodiment, the engineered microorganisms with partial orcomplete loss of ADH and/or alkene reductase activity have improvedtolerance to crotonaldehyde and/or crotyl alcohol. In some embodimentsthe engineered microorganisms include genetic modifications causing fullor partial loss of ADH and/or alkene reductase activity and geneticmodifications that include crotyl alcohol and/or other pathways that usecrotyl alcohol as an intermediate. These various embodiments can beassessed by assaying their growth in crotyl alcohol concentrations thatare detrimental to growth of the parental strains. Tolerance to crotylalcohol may vary depending on the crotyl alcohol concentration, growthconditions and the specific engineered strain. For example, as shown inExample 2, a deletion in adh and/or nemA showed improved growth over theparental strain without the modification.

The engineered microorganisms and methods can be described in terms oftheir growth in media including crotyl alcohol. Growth can optionally bedefined in terms of other parameters such as other media components, thetemperature and other conditions in which the cells are grown. It isunderstood that during culturing, media conditions will constantlychange as caused by consumption and depletion of media nutrients by thecells, and the increase in the amount bioproducts in the media such ascrotyl alcohol. Accordingly, the cell's growth rate may change atcertain points during culturing. Growth and growth rate of cells canoptionally be described in a certain amount of a media component, or acertain range of a media component.

Culturing includes growing (proliferation phase) or maintaining themicroorganism at a stationary phase. Production of crotyl alcohol or itsdownstream products can occur in either or both phases

In some embodiments, growth of the engineered microbial cell isdescribed in terms of its capability of growing in media containing anamount of crotyl alcohol, where its growth is measured relative to aparental strain that does not have genetic modifications to one or moregenes that provides tolerance to crotyl alcohol, and/or increases crotylproduction. For example, deletions in genes that encode for ADH and/oralkene reductase activity exhibit an increase in the growth rate inmedia of 1.1 times or greater as compared to the growth rate of a parenthost strain or microorganism that does not have the referenced geneticmodifications. The increase in the rate of growth can be measured inmedia containing 0.001 to about 2 wt % of crotyl alcohol. For example,the engineered microorganism can exhibit a growth rate of 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, and 2.5 times that ofthe parent host strain without the genetic modification. In otherembodiments, the engineered microorganism can have a grow rate of about1.1 times to about 3 times, or about 1.2 times to about 2.5 times thatof the parent host strain without the genetic modification.

In some embodiments, the engineered microorganism is grown in culturehaving more than 0.001% (w/v) of crotyl alcohol, or more than 0.01%,0.1%, 0.5%, 1% or 2% (w/v) of crotyl alcohol.

In some embodiments, the engineered microorganism is grown in culturehaving more than 0.138 mM of crotyl alcohol, or more than 1.38 mM 13.8mM, 69 mM, 138 mM or 276 mM of crotyl alcohol.

In one embodiment, suitable ADH and/or alkene reductase enzymes catalyzethe reactions optimally at host cell physiological conditions. Inanother embodiment, suitable ADH and/or aldehyde reductase enzymes foruse catalyze reactions optimally from about pH 4 to about pH 9. Inanother embodiment, suitable ADH and/or alkene reductase enzymescatalyze reactions optimally from about pH 5 to about pH 8. In anotherembodiment, suitable ADH and/or alkene reductase enzymes for usecatalyze reduction reactions optimally from about pH 6 to about pH 7. Inanother embodiment, suitable ADH and/or alkene reductase enzymescatalyze reactions optimally from about pH 6.5 to about pH 7. In anotherembodiment, suitable ADH and/or alkene reductase enzymes for usecatalyze reactions optimally at about pH 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, or 9. In another embodiment, suitable ADH and/or alkenereductase enzymes for use catalyze reactions optimally at about pH 7.

In one embodiment, suitable ADH and/or alkene reductase enzymes for usecatalyze reactions optimally at up to about 70° C. In anotherembodiment, suitable ADH and/or alkene enzymes catalyze reductionreactions optimally at about 10° C., 15° C., 20° C., 25° C., 30° C., 35°C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C. In anotherembodiment, suitable ADH and/or alkene reductase enzymes catalyzereactions optimally at about 30° C.

EXAMPLES Example 1 Strain Construction/Deletion Methods

Alcohol dehydrogenases; adhE (GI: 732684842), yghD (GI: 16130909), adhP(GI: 732684610), yahK (GI:1657523) and N-ethylmaleimide reductase, nemA(GI: 732684441) were deleted from E. coli ATCC 8739 C variant and E.coli Str. K-12 substr. MG1655. Strains were constructed using A, redrecombination as described in Datsenko and Wanner, Proc Natl Acad SciUSA (2000), 97(12): 6640-6645. Briefly, strains were transformed withpRED plasmid containing arabinose inducible λ red recombinase genes anda bla resistance cassette conferring carbenicillin resistance.Transformants carrying the pRED plasmid were then grown in 100 μg/mLcarbenicillin and 134 mM arabinose and transformed with linear DNAconstructs containing homology arms targeting the gene to be deletedflanking a sacB-kan cassette containing constitutively expressed sacBand kan genes. The kan gene confers kanamycin resistance and allows forselection of cells with integrated sacB-kan cassettes when grown on 50μg/mL kanamycin. The sacB gene confers sucrose sensitivity and allows acounter selective pressure to select for cells that no longer have theintegrated sacB-kan cassette. Cells were grown in the presence of 100μg/ml, carbenicillin and 50 μg/mL kanamycin to confirm integration ofsacB-kan cassette and presence of plied plasmid. Cells were then inducedwith 134 mM arabinose and transformed with a linear DNA constructreplacing the sacB-kan cassette with DNA homologous to chromosomal DNAflanking the desired deletion, effectively deleting the desired DNAsequence (see Table 5 for deleted DNA sequences for each strain).Replacement of the sacB-kan cassette was selected for by growth in 303mM sucrose. Chromosomal deletion was confirmed by PCR amplification andsequencing (see Table 5 for primers used for amplification andsequencing).

In this example, for deletion of more than one gene in an organism, thestrain with a first deletion was constructed and used for eachsubsequent gene deletion.

TABLE 5 DNA Sequences deleted from chromosome and primers used andreported in the Sequence Listing. Chromosomal Modification Sequence IDNo. adhE deletion SEQ ID NO.: 1 yqhD deletion SEQ ID NO.: 2 adhPdeletion SEQ ID NO.: 3 yahK deletion SEQ ID NO. 4 nemA deletion SEQ IDNO.: 5 adhEF1 SEQ ID NO. 6 adhER1 SEQ ID NO. 7 yqhDF1 SEQ ID NO. 8yqhDR1 SEQ ID NO. 9 adhPF1 SEQ ID NO. 10 adhPR1 SEQ ID NO. 11 yahKF1 SEQID NO. 12 yahKR1 SEQ ID NO. 13 nemAf1 SEQ ID NO. 14 nemAR1 SEQ ID NO. 15adhE protein SEQ ID NO. 16 yqhD protein SEQ ID NO. 17 adhP protein SEQID NO. 18 yahK protein SEQ ID NO. 19 nemA protein SEQ ID NO. 20

Example 2 Crotyl Alcohol Tolerance Studies

E. coli Str. K-12 substr. MG1655 strains 48 (wildtype) and 3-FEDB withmultiple deletions (ΔyahkΔadhPΔyqhDΔadhE); and E. coli ATCC 8739 Cstrains (wildtype) and its variants 3-FISC (ΔadhE), 3-GASB (ΔadhEΔyqhD),3-GROG (ΔadhEΔyqhDΔadhP), and 3-GDOB (ΔadhEΔyqhDΔadhPΔyahK) were struckfor single colonies on LB agar from a glycerol freezer stock. Foursingle colonies per strain were picked and inoculated into 1 mL LB+55.7mM glucose in deep well 96 well plates and grown at 37° C. overnight(16-20 hours). The optical density of the overnight culture was takenand cells were normalized to OD₆₀₀=1 and spun down. Cells wereresuspended in 1 mL of M9 minimal media+11.1 mM glucose. Cells were thenspun down and washed again in 1 mL M9+11.1 mM glucose. Bioscreenhoneycomb 2 plates (Cat. No. 9502550) were filled with 198 μL M9+11.1 mMglucose or M9+11.1 mM glucose+138 mM crotyl alcohol (Sigma-Aldrich). 2μL washed cells were added to each well at N=4. Bioscreen honeycomb 2plates were loaded into a bioscreen C MBR (Oy Growth Curves Ab Ltd)machine, an incubator system set to read turbidity at OD₄₂₀₋₅₈₀ every 15minutes at 37° C. For growth curve data, see FIG. 4A (E. coli Str. K-12substr. MG1655 strains) and FIG. 4B (K coil ATCC 8739 C variantstrains).

FIG. 4A shows that when grown in the absence of crotyl alcohol, thewild-type or parental strain and the engineered microorganisms showsimilar growth rates. FIG. 4B shows that when grown in the presence of138 mM crotyl alcohol, the wild-type or parental strain's growth isinhibited, whereas the engineered microorganism 3-FEDB(ΔyahKΔadhPΔyqhDΔadhE) while showing an initial lag grew on crotylalcohol.

FIG. 5A shows that when grown in the absence of crotyl alcohol, thewild-type or parental strain and the engineered microorganisms showsimilar, irregular growth rates. FIG. 5B shows that when grown in thepresence of 138 mM crotyl alcohol, the wild-type or parental strain'sgrowth is inhibited as are the 3-FIDC (ΔadhE) and 3-GAEB (ΔadhEΔyqhD),whereas the engineered microorganisms 3-GBOG (ΔyahKΔadhPΔyqhDΔadhE) and3-GBOB (ΔadhEΔyqhDΔadhP) while showing an initial lag grew on crotylalcohol.

Example 3 NemA Validation Experiments

Strains (ATCC 8739 C variants) 3-GGGF (yqhD+nemA+ parent strain),3-01100 (ΔyqhD), 3-HAEF (ΔnanA), and 3-HAEG (ΔyqhDΔnemA) were struckonto LB agar plates from glycerol stock and grown overnight (16-20hours). Single colonies were picked and inoculated into 30 mL M9+27.8 mMglucose+10 mM crotyl alcohol in baffled flasks and grown overnight(16-20 hours). Overnight cultures were seeded at OD₆₀₀=0.1 into fresh 30mL M9+55.7 mM glucose+10 mM crotyl alcohol in baffled flasks. 1 mLsamples were taken at 4, 8, and 27 hours after growth on crotyl alcohol.Samples were spun down, and the supernatants were submitted foranalytical analysis. GCMS analysis of crotyl alcohol and n-butanol inliquid samples by liquid injection. Reduction of butanol was quantifiedin GCMS chromatograms and line graphs.

GCMS Methods

A GCMS method has been developed monitor the following volatilecompounds: n-butanol (n-ButOH) and crotyl alcohol (Crot-OH). This methodhas good sensitivity with detection limits in low mM range (linearity in0.5-100 mM). Sensitivity of this assay is compound dependent, being afunction of volatility and partial pressures under the conditions usedand MS ionization/fragmentation efficiency. Crotyl alcohol and n-butanolstandards were purchased from Sigma-Aldrich.

GCMS Analysis

An Agilent gas chromatograph 6890N, interfaced to a mass-selectivedetector (MSD) 5973, operated in electron impact ionization (EI) mode,was used for the analysis. An INNOWAX column (Agilent 19091N-133), 30m×0.25 mm i.d.×0.25 μm film thickness, was chosen. Samples were injectedby an ALS autosampler operated in direct injection mode. The parametersused were: injection volume 1.0 μL (split ratio 20:1), 250° C. inlettemperature. Helium was used as a carrier gas, and the flow ratemaintained at 1.3 mL/min. A temperature program has been optimized toensure good resolution of the analytes of interest and minimum matrixinterference: the oven is initially held at 40° C. for 1 min, thenramped to 41° C. at 1° C./min, second ramp at 35° C./min to 60° C., andfinal ramp at 100° C./min to 250° C. and hold 4.06 min (total run time10 min). The data were acquired using low mass. MS tune file settingsand 28-150 m/z mass-range scan. Typical retention times (RT) andcharacteristic mass fragments used for quantitation of the analytes ofinterest are listed in Table 6. A typical GCMS chromatogram of astandard mix is presented in the figures referred to below. Quantitationwas performed based on standard curves using quadratic fit (typically,forced to zero). GCMS data were processed using Agilent MSD ProductivityChemStation software.

TABLE 6 Analyte RT, min Target m/z n-propanol (IS) 4.96 59 n-butanol5.90 56 Trans-crotyl alcohol 6.29 72 Cis-crotyl alcohol 6.40 72

The engineered microorganisms 3-GHDO (ΔyqhD), 3-HAEF (Mewl), and 3-HAEGwith multiple deletions (ΔnemAΔyghD) exhibited no detectable butanolproduction whereas they exhibit crotyl alcohol and propanol production,as reported in FIG. 6A-6D based on GC/NIS chromatographs of organismsgrown on crotyl alcohol-containing media and assayed for n-propanol,butanol and crotyl alcohol for 4, 8 and 27 hours.

1-33. (canceled)
 34. A method for producing crotyl alcohol using anengineered microorganism, where crotyl alcohol is a bioproduct, orcrotyl alcohol is an intermediate in the bioproduct pathway, the methodcomprising: culturing the engineered microorganism under conditionsresulting in crotyl alcohol or use of crotyl alcohol as the intermediatein the bioproduct pathway resulting in bioproduct, wherein theengineered microorganism comprises a genetic modification causingpartial or complete loss of activity in an alkene reductase thatconverts crotonaldehyde to butyraldehyde and at least one exogenousnucleic acid encoding an enzyme of the bioproduct pathway, where thebioproduct pathway is a crotyl alcohol pathway or uses a crotyl alcoholas an intermediate in the bioproduct pathway.
 35. The method of claim34, wherein the engineered microorganism further comprises at least onegenetic modification causing partial or complete loss of activity in (a)an alcohol dehydrogenase (ADH) that converts crotyl alcohol tocrotonaldehyde, or (b) an ADH that converts butyraldehyde to butanol, or(c) both combination of (a) and (b).
 36. The method of claim 35, whereinthe ADH activity is encoded by a gene selected from adhE, adhP, yqhD andyahK or any combination thereof.
 37. The method of claim 34, wherein thealkene reductase activity is selected from EC Number: 1.3.1.-class. 38.The method of claim 34, wherein the alkene reductase activity is encodedby a nemA gene or yqjM gene or both.
 39. The method of claim 34, whereinthe partial or complete loss of alkene reductase reduces theaccumulation or conversion of butyraldehyde or butanol or bothbutyraldehyde and butanol.
 40. The method of claim 34, wherein thecrotyl alcohol bioproduct pathway comprises an acetyl-CoA:acetyl-CoAacyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoAdehydratase, a crotonyl-CoA reductase (aldehyde forming), acrotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase,synthetase, or transferase, a crotonate reductase, a crotonyl-CoAreductase (alcohol forming), a glutaconyl-CoA decarboxylase, aglutaryl-CoA dehydrogenase, a 3-aminobutyryl-CoA deaminase, or a4-hydroxybutyryl-CoA dehydratase.
 41. The method of claim 34, whereinthe engineered microorganism comprises an enzyme converting crotylalcohol to 1,3-butadiene, or where the enzyme is a linalool dehydratase,and/or wherein the crotyl alcohol produced is converted to 3-buten-2-oland/or butadiene.
 42. The method of claim 34, wherein the engineeredmicroorganism comprises a crotyl alcohol to butadiene pathway comprisingone or more of the following exogenous nucleic acids encoding enzymes:crotyl alcohol kinase, 2-butenyl-4-phosphate kinase, butadiene synthase,3-buten-2-ol synthase, 3-buten-2-ol synthase, crotyl alcohol isomerase,3-buten-2-ol dehydratase, crotyl alcohol dehydratase, crotyl alcoholdiphosphokinase, crotyl alcohol dehydratase/vinyl isomerase.
 43. Themethod of claim 34, wherein the crotyl alcohol produced is converted to3-buten-2-ol and/or butadiene.
 44. The method of claim 34, wherein theengineered microorganism exhibits an increased growth on crotyl alcoholcompared to a parental host microorganism that does not have the geneticmodifications.
 45. The method of claim 34, wherein the engineeredmicroorganism grows on medium containing about 0.138 mM to about 138 mMcrotyl alcohol.
 46. A cell culture composition comprising crotyl alcoholor butadiene produced by the method of claim
 34. 47. An engineeredmicroorganism comprising at least one genetic modification causingpartial or complete loss of activity in an alkene reductase thatconverts crotonaldehyde to butyraldehyde and an exogenous nucleic acidencoding an enzyme of a bioproduct pathway, where the bioproduct pathwayis a crotyl alcohol pathway or uses a crotyl alcohol as an intermediatein the bioproduct pathway.
 40. The engineered microorganism of claim 47,wherein the engineered microorganism further comprises at least onegenetic modification causing partial or complete loss of activity in (a)an alcohol dehydrogenase (ADH) that converts crotyl alcohol tocrotonaldehyde, or (b) an ADH that converts butyraldehyde to butanol, or(c) both combination of (a) and (b).
 48. The engineered microorganism ofclaim 47, wherein the ADH activity is encoded by a gene selected fromadhE, adhP, yqhD and yahK or any combination thereof.
 50. The engineeredmicroorganism of claim 47, wherein the alkene reductase activity isencoded by a nemA gene or yqjM gene or both.
 51. The engineeredmicroorganism of claim 47, wherein the engineered microorganism exhibitsan increased growth on crotyl alcohol compared to a parental hostmicroorganism that does not have the genetic modifications.
 52. Theengineered microorganism of claim 47, wherein the engineeredmicroorganism grows on medium containing about 0.138 mM to about 138 mMcrotyl alcohol.
 53. The engineered microorganism of claim 47, whereinthe crotyl alcohol bioproduct pathway comprises an acetyl-CoA:acetyl-CoAacyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoAdehydratase, a crotonyl-CoA reductase (aldehyde forming), acrotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase,synthetase, or transferase, a crotonate reductase, a crotonyl-CoAreductase (alcohol forming), a glutaconyl-CoA decarboxylase, aglutaryl-CoA dehydrogenase, a 3-aminobutyryl-CoA deaminase, or a4-hydroxybutyryl-CoA dehydratase.